The vitamin K-dependent gamma-glutamyl carboxylase post-translationally converts glutamic acids in its substrate proteins to gamma carboxyglutamic acid (Gla). This modification is critical for normal blood coagulation, through multiple vitamin K-dependent proteins, and for calcium homeostasis, through participation of matrix Gla protein. It is likely that other critical roles for this post-translational modification will be discovered. Here we explore the structure and function of the carboxylase. We have been shown that the ability of the carboxylase to activate vitamin K to the highly reactive cofactor intermediate is regulated by the presence of glutamate containing substrate. This regulation results from modification, at least in part, of the environment of a critical free cysteine residue in the enzyme. In specific Aim 1 we identify the mechanistically important cysteine(s) and determine the disulfide bonding pattern in carboxylase. In Specific Aim 2, we determine the topology of the carboxylase within the endoplasmic reticulum membrane. We will determine the potential N-linked glycosylation sites in the enzyme that are occupied, perform glycosylation scanning mutagenesis and use cell free transcription and translation in conjunction with protease protection assays. In Specific Aim 3 we use innovative methods of tandem mass spectrometry that spares gamma-carboxyl groups during ion fragmentation to determine if the carboxylase is processive or distributive, if distributive, is it directional or random? We developed a transgenic mouse model in which, on the background of a carboxylase null mouse, we introduced a wild-type carboxylase transgene under the regulation of a liver specific promoter. This rescues the fetal phenotype of the carboxylase null mouse. In Specific Aim 4 we will use this model to identify additional critical gamma-carboxylated proteins. We will also use the mouse model to determine the relationship of kinetic parameters of various carboxylase substrates to in vivo carboxylation of these substrates. The vitamin K-dependent carboxylase performs a post-translational modification critical for blood coagulation and for calcium homeostasis. The studies proposed will elucidate important aspects of carboxylase mechanism and provide a topologic structure to unify the mechanistic information already available for this enzyme. The performance of these studies will be enhanced by other aspects of the Program: the availability of Core services, information about regulation of carboxylase gene expression from Project II as well as the technical expertise of Drs. David Roth and Bruce Furie in various aspects of these studies.